Consumable Liquid Effects
on Amylase Activity
By: Pat Ebbert
Pittsburgh Central Catholic
PJAS 2009
Introduction
 Amylase is an enzyme found in the body that
breaks down starch into simple sugars.
 Amylase is found in the saliva and in the
pancreas of humans, breaking down the
starch from the foods we consume into
simpler sugars, which are later converted into
glucose needed for energy.
Alpha-Amylase
 Found in human saliva
and pancreas.
 Breaks down long
strands of
carbohydrates into
simpler sugars such as
maltose or glucose.
 Considered superior to
β-Amylase because it
can act on any portion
of the substrate making
reactions occur much
faster.
Structure of Alpha-Amylase
 To the right is a diagram
showing the structure of
the Alpha-Amylase
enzyme.
 The green dot
represents a chloride
ion, the pale yellow a
calcium ion. Both are
used to help the
enzyme bind to the
substrate.
What affects Amylase?
 Is amylase activity affected by the presence
of consumable liquids in the oral cavity?
Variable #1: Diet Pepsi
 Diet Pepsi contains no
sugar.
 Ingredients: Carbonated
water, aspartame,
phosphoric acid,
potassium benzoate,
caffeine, citric acid
Variable #2: Mylanta Antacid
 Mylanta Antacid is used
to neutralize stomach
acid that leads to upset
stomach and nausea.
 Ingredients: Aluminum
hydroxide, magnesium
hydroxide, simethicone.
Variable #3: Listerine
 Listerine is made to kill
bacteria in the oral
cavity that cause plaque
buildup and bad breath.
 Ingredients: water,
menthol, methyl
salicylate, eucalyptol,
thymol, alcohol, benzoic
acid, sorbitol, sodium
benzoate
Purpose
 To determine the effect of common
consumable liquids on salivary amylase
activity.
Hypothesis
 Null: The presence of Mylanta, Diet Pepsi,
and Listerine will each have NO significant
effect on the activity of enzyme alphaamylase.
 Alternative: The presence of Mylanta, Diet
Pepsi, and Listerine WILL each have a
significant effect on the activity of enzyme
alpha-amylase.
Materials
 2% stock solution of 1,4-





α-D-glucan
glucanohydrolase
(alpha-amylase) in
distilled water
3% stock solution of
iodine in distilled water
5ml macro-pipette
50-200µl micro-pipette
Micro-pipette tips
16 starch-agar plates
 8 13x100mm boro






silicate test tubes
50ml conical plastic
tube
Squirt bottle
Sharpie marker
Drinking straws
Diet Pepsi
Mylanta antacid drink
Listerine mouthwash
Important Property of Iodine
 Iodine has the property of
An iodine atom
binding to starch to create a
dark blue color.
 If starch has been broken down
into simpler sugars, the iodine
cannot bind and therefore
leaves an area of no color
visible.
 If amylase has broken down
surrounding starch into simpler
sugars, this area’s diameter can
be measured to give a relative
value of how much starch has
been broken down.
General Procedure
1.
2.
3.
4.
Eight different test tube solutions were created to
be used in experiment (explained on following
slide).
Five wells in each of 16 starch agar plates were
filled with 50µl of amylase for eight different data
sets (10 trials of each set, 80 total wells).
Two data sets were positive and negative control
sets, three were 10% variable solutions, and three
were 50% variable solutions (all of these came
from the test tubes).
After one hour, iodine solution was sprayed onto
each plate and zone of starch-breakdown was
measured in millimeters.
General Procedure (cont.)
Neg. Control
Pos. Control
10% Var.*
50% Var.*
Amylase
solution
0ml
1ml
1ml
1ml
Distilled water
5ml
4ml
3.5ml
1.5ml
Variable
0ml
0ml
0.5ml
2.5ml
Total
5ml
5ml
5ml
5ml
This table represents what was present in the eight test tubes used in
the experiment. After the solutions were prepared, 50µl of each tube
were transferred into ten different starch-agar plate wells.
*The variable columns were repeated three times (Mylanta, Diet Pepsi,
Listerine)
Consumable Liquid Effects on Amylase Activity
P-value received from single-factor Anova= 2.76 x10-29
16
Avg. Zone of Starch Digestion
in mm
14
P-values for all variables
less than .01 found using
Dunnett’s Test
12
10
8
6
4
2
0
Pos. Con.
Neg. Con.
DP10
DP50
MY10
Consumable Liquids
MY50
LIS10
LIS50
ANOVA Statistical Analysis
ANOVA
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
432.2857
6
72.04762
49.2
63
0.780952
481.4857
69
F
P-value
92.2561
2.76E-29
F crit
2.246408
Dunnett’s Test Results
α = .01, t-crit = 4.08
Variable compared to
Positive Control
(14.6mm avg.)
t-value
Interpretation
Diet Pepsi 10%
(11.9mm avg.)
6.83
Significant Variance
Diet Pepsi 50%
(8.8mm avg.)
14.67
Significant Variance
Mylanta 10%
(10.5mm avg.)
10.37
Significant Variance
Mylanta 50%
(6.5mm avg.)
20.49
Significant Variance
Listerine 10%
(10.4mm avg.)
10.63
Significant Variance
Listerine 50%
(7.9mm avg.)
16.95
Significant Variance
Results and Conclusions
 The null hypothesis that the presence of
Mylanta, Diet Pepsi, and Listerine will each
have NO significant effect on the amylase
activity was rejected (p-value < .05).
 The alternative hypothesis that the presence
of Mylanta, Diet Pepsi, and Listerine WILL
each have a significant effect on the amylase
activity was therefore accepted.
Limitations and Extensions
 Sources of Error,
Limitations
 The amylase chemicals
could have degraded over
time, yielding less effective
binding to the starch.
 While using straws to create
wells, extra salivary amylase
may have entered the plates,
increasing starch
degradation.
 Measurements taken with
the naked eye could be
slightly off.
 Extensions
 To increase accuracy, the
amylase could have been
given more time to react with
the starch-agar.
 Also, more trials are always
useful for more accurate
results.
 Precise measurements could
have been used to ensure
that the data was recorded
correctly.
References
 “Alpha-Amylase”. Wikipedia.org. <http://en.wikipedia.org/wiki/Alpha





Amylase>. December 14, 2008.
“Antacids”. eMedTV.com.
<http://gerd.emedtv.com/antacids/antacids.html>. December 14, 2008.
Bassiouny, M.A.; Yang, J. "Influence of drinking patterns of carbonated
beverages on dental erosion”. General Dentistry, May-June, vol. 53, no.
3, 2005.
“Comparing Means with a Control”. Davidmlane.com.
<http://davidmlane.com/hyperstat/B112114.html>. January 17, 2009.
“Listerine”. Wikipedia.org. <http://en.wikipedia.org/wiki/Listerine>.
December 14, 2008.
“Uses of Iodine”. Pleasantridge.k12.ca.us.
<http://www.pleasantridge.k12.ca.us/magnolia/elements/iodine/iodine2.
html>. December 14, 2008.
Wang, Nam Sun. “Starch Hydrolysis by Amylase”.
<http://www.eng.umd.edu/~nsw/ench485/lab5.htm>. December 20,
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